Iap binding peptides and assays for identifying compounds that bind iap

ABSTRACT

Assays are disclosed for identifying peptides and peptidomimetics for promoting apotosis in cells, through a pathway involving the Inhibitor of Apoptosis Proteins (IAPs), exemplified by XIAP, and the mitochondrial protein Smac/DIABOLO (hereinafter Smac) and homologs thereof. Also disclosed are IAP-binding peptides and peptidomimetics identified through the use of the assay.

This application claims benefit of U.S. Provisional Application Nos.60/294,682, filed May 31, 2001, and 60/345,630, filed Jan. 3, 2002, theentirety of each of which is incorporated by reference herein.

Pursuant to 35 U.S.C. §202(c), it is acknowledged that the U.S.Government has certain rights in the invention described herein, whichwas made in part with funds from the National Institutes of Health,Grant No. GM59348-02.

FIELD OF THE INVENTION

The present invention relates to the field of drug design anddevelopment for prevention and treatment of cell proliferative disease.Specifically, the invention features an assay for identifying peptidesand peptidomimetics for promoting apotosis in cells, through a pathwayinvolving the Inhibitor of Apoptosis Proteins (IAPs), exemplified byXIAP, and the mitochondrial protein Smac/DIABOLO (hereinafter Smac). Theinvention also features peptides and peptidomimetics identified throughthe use of the assay.

BACKGROUND OF THE INVENTION

Various scientific articles, patents and other publications are referredto throughout the specification. Each of these publications isincorporated by reference herein in its entirety.

Apoptosis (programmed cell death) plays a central role in thedevelopment and homeostasis of all multi-cellular organisms. Alterationsin apoptotic pathways have been implicated in many types of humanpathologies, including developmental disorders, cancer, autoimmunediseases, as well as neuro-degenerative disorders.

Thus, the programmed cell death pathways have become attractive targetsfor development of therapeutic agents. In particular, since it isconceptually easier to kill than to sustain cells, attention has beenfocused on anti-cancer therapies using pro-apoptotic agents such asconventional radiation and chemo-therapy. These treatments are generallybelieved to trigger activation of the mitochondria-mediated apoptoticpathways. However, these therapies lack molecular specificity, and morespecific molecular targets are needed.

Apoptosis is executed primarily by activated caspases, a family ofcysteine proteases with aspartate specificity in their substrates.Caspases are produced in cells as catalytically inactive zymogens andmust be proteolytically processed to become active proteases duringapoptosis. In normal surviving cells that have not received an apoptoticstimulus, most caspases remain inactive. Even if some caspases areaberrantly activated, their proteolytic activity can be fully inhibitedby a family of evolutionarily conserved proteins called IAPs (inhibitorsof apoptosis proteins) (Deveraux & Reed, Genes Dev. 13: 239-252, 1999).Each of the IAPs contains 1-3 copies of the so-called BIR (baculoviralIAP repeat) domain and directly interacts with and inhibits theenzymatic activity of mature caspases. Several distinct mammalian IAPsincluding XIAP, survivin, and Livin/ML-IAP (Kasof & Gomes, J. Biol.Chem. 276: 3238-3246, 2001; Vucic et al. Curr. Biol. 10: 1359-1366,2000; Ashhab et al. FEBS Lett. 495: 56-60, 2001), have been identified,and they all exhibit anti-apoptotic activity in cell culture (Deveraux &Reed, 1999, supra). As IAPs are expressed in most cancer cells, they maydirectly contribute to tumor progression and subsequent resistance todrug treatment.

In normal cells signaled to undergo apoptosis, however, the LAP-mediatedinhibitory effect must be removed, a process at least in part performedby a mitochondrial protein named Smac (second mitochondria-derivedactivator of caspases; Du et al. Cell 102: 33-42, 2000) or DIABLO(direct IAP binding protein with low pI; Verhagen et al. Cell 102:43-53, 2000). Smac, synthesized in the cytoplasm, is targeted to theinter-membrane space of mitochondria. Upon apoptotic stimuli, Smac isreleased from mitochondria back into the cytosol, together withcytochrome c. Whereas cytochrome c induces multimerization of Apaf-1 toactivate procaspase-9 and -3, Smac eliminates the inhibitory effect ofmultiple IAPs. Smac interacts with all IAPs that have been examined todate, including XIAP, c-IAP1, c-IAP2, and survivin (Du et al., 2000,supra; Verhagen et al., 2000, supra). Thus, Smac appears to be a masterregulator of apoptosis in mammals.

Smac is synthesized as a precursor molecule of 239 amino acids; theN-terminal 55 residues serve as the mitochondria targeting sequence thatis removed after import (Du et al., 2000, supra). The mature form ofSmac contains 184 amino acids and behaves as an oligomer in solution (Duet al., 2000, supra). Smac and various fragments thereof have beenproposed for use as targets for identification of therapeutic agents.U.S. Pat. No. 6,110,691 to Wang et al. describes the Smac polypeptideand fragments ranging from at least 8 amino acid residues in length.However, the patent neither discloses nor teaches a structural basis forchoosing a particular peptide fragment of Smac for use as a therapeuticagent or target.

Similar to mammals, flies contain two IAPs, DLAP1 and DIAP2, that bindand inactivate several Drosophila caspases (Hay, Cell Death Differ. 7:1045-1056, 2000). DIAP1 contains two BIR domains; the second BIR domain(BIR2) is necessary and sufficient to block cell death in many contexts.In Drosophila cells, the anti-death function of DIAP1 is removed bythree pro-apoptotic proteins, Hid, Grim, and Reaper, which physicallyinteract with the BIR2 domain of DIAP1 and remove its inhibitory effecton caspases. Thus Hid, Grim, and Reaper represent the functionalhomologs of the mammalian protein Smac. However, except for theirN-terminal 10 residues, Hid, Grim, and Reaper share no sequence homologywith one another, and there is no apparent homology between the threeDrosophila proteins and Smac.

In commonly-owned co-pending application Ser. No. 09/965,967 (theentirety of which is incorporated by reference herein), it is disclosedthat the above described biological activity of Smac is related tobinding of its N-terminal four residues to a featured surface groove ina portion of XIAP referred to as the BIR3 domain. This binding preventsXIAP from exerting its apoptosis-suppressing function in the cell. Itwas further disclosed that N-terminal tetrapeptides from LAP bindingproteins of the Drosophila pro-apoptotic proteins Hid, Grim and Vetofunction in the same manner.

The development of apoptosis-promoting therapeutic agents based on theIAP-binding peptide of Smac or its homologs from other species would begreatly facilitated by high throughput screening assays to identifyuseful molecules. Further, development of such therapeutic agents wouldbe accelerated by the production of libraries of rationally designedcandidate compounds.

SUMMARY OF THE INVENTION

The present invention features an assay for use in high throughputscreening or rational drug design of agents that can, like the Smactetrapeptide or its homologs in other species, bind to a BIR domain ofan IAP, thereby relieving IAP-mediated suppression of apoptosis. Theseassays make use of the discoveries made in accordance with the inventiondisclosed in commonly-owned, co-pending U.S. application Ser. No.09/965,967 that (1) the N-terminal tetrapeptide motif of Smac and otherIAP binding proteins is sufficient for binding to IAPs and (2) themammalian BIR3 domain and the Drosophila BIR2 domain comprise a specificbinding groove for the tetrapeptide.

The assay comprises the following basic steps: (a) providing a labeledmimetic of an IAP-binding tetrapeptide that binds to the appropriate BIRdomain (preferably BIR3), wherein at least one measurable feature of thelabel changes as a function of the mimetic being bound to the IAP orfree in solution; (b) contacting the BIR domain of an IAP with thelabeled mimetic under conditions enabling binding of the mimetic to theBIR domain, thereby forming a BIR-labeled mimetic complex having themeasurable feature; (c) contacting the BIR-labeled mimetic complex withthe compound to be tested for BIR binding; and (d) measuringdisplacement of the labeled mimetic from the BIR-labeled mimeticcomplex, if any, by the test compound, by measuring the change in themeasurable feature of the labeled mimetic, thereby determining if thetest compound is capable of binding to the LAP. In a preferredembodiment, the labeled mimetic is AVPX (SEQ ID NO:1), wherein X isdirectly or indirectly linked to a fluorigenic dye. Preferably, it isAVPC (SEQ ID NO:2) attached to a badan dye.

The present invention also provides a library of peptides orpeptidomimetics that have been demonstrated by the methods of theinvention to bind to the BIR3 domain of XIAP. In one embodiment, thesepeptides are composed of naturally-occurring amino acid residues. Inanother embodiment, the library is based on a peptidomimetic, which maybe partially or fully non-peptide in nature, but which mimics thephysicochemical features of the Smac peptide such that it is capable ofbinding IAP.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the chemical structure of AVPC-badan dye.

FIG. 2 shows absorption and emission properties of AVPC-badan. FIG. 2Ashows the absorption (solid line) and emission (dotted line) spectra ofthe molecule in water. FIG. 2B shows the solvatochromicity of AVPC-badanin acetonitrile (ACN), with respect to the emission spectrum.

FIG. 3 shows the emission spectra of AVPC-badan in the presence of BIR3at different concentrations of BIR3. Measurements were taken in 50 mMTris buffer, pH 7.1, 100 mM NaCL, 2 mM DTT and 5.1 μM badan dye,excitation wavelength 387 nm.

FIG. 4 shows emission spectra of samples from the binding assaydescribed in the text, the results of which are shown in Table 2. Allsamples were 5 μM in both dye and protein, and 50 mM in thetetrapeptide. The buffer was 50 mM Tris at pH 7.1, 100 mM NaCl and 2 mMDTT. The AVPI (SEQ ID NO:3) tetrapeptide displayed was synthesizedseparately from the other samples.

FIG. 5 shows (A) absorption (−) and emission ( - - - ) spectra ofAVPC-badan in water (excitation at 387 nm) (These spectra are also shownin FIG. 2); and (B) titration of AVPC-badan with BIR3. The fraction offree AVPC-badan was determined by relating the difference of theobserved fluorescence intensity and a maximum intensity where all of thedye is assumed to be bound, I_(v), to the difference between theintensity of the unbound dye and I_(v). Data are discussed in Example 1.

FIG. 6 shows (A) emission spectra of AVPC-badan, AVPC-badan in thepresence of BIR3 and AVPF (SEQ ID NO:4), AVPC-badan in the presence ofBIR3 and ARPI (SEQ ID NO:5), AVPC-badan in the presence of BIR3 and AVPI(SEQ ID NO:3), AVPC-badan in the presence of BIR3 and GVPI (SEQ IDNO:6), AVPC-badan in the presence of BIR3 and AGPI (SEQ ID NO:7), andAVPC-badan in the presence of BIR3, in order of increasing emissionintensity; and (B) correlation of hydrophobic interaction expressed asΔG₁ (EtOH—H₂O) (23) with ΔG_(b) for a range of nonpolar amino acids(polar amino acids are not shown in this graph). Data are discussed inExample 1.

DETAILED DESCRIPTION OF THE INVENTION

The ability to quickly assay small molecules for their effectiveness indisrupting protein-protein interactions is critical to the developmentof viable drug candidates. One aspect of the present invention comprisesan assay to test the binding affinity of a library of tetrapeptidemolecules for the BIR3 domain of an inhibitor of apoptosis protein(LAP), particularly the mammalian XIAP. The assay is based on adetectable label, preferably a fluorogenic dye molecule. In preferredembodiments, the fluorophore is attached to a tripeptide, AVP, whosesequence matches the N-terminal three residues of Smac. The generalstructure of this molecule, therefore, is AVP[X], wherein X is thefluorophore. The molecule is referred to herein as an “AVP-dye”. TheAVP-dye packs into the groove of the BIR3, causing a large shift inemission maximum and intensity when the environment of the dye changesfrom water to the hydrophobic pocket of the protein. If a molecule (e.g.the native Smac protein or a tetrapeptide mimic) displaces the dye, thenemission will shift back to the spectrum observed in water. Since theemission intensity is related to the binding of the tetrapeptide, theintensity can be used to estimate the equilibrium constant, K, fordisplacement of the AVP-dye by the tetrapeptide. The larger theequilibrium constant, the greater affinity the tetrapeptide has for theBIR3. This allows the most promising inhibitors to be quicklydetermined, and structural information about effective inhibitors can beincorporated into the design of candidates for the next round oftesting.

It will be understood by those of skill in the art that, though the AVPdye-BIR3 system described above is exemplified and preferred forpractice of the invention, various combinations of (1) LAP-bindingtetrapeptides and mimetics, (2) BIR binding grooves and (3) detectablelabels may be used interchangeably to create variations of the assaydescribed above. Particular reference is given to the consensustetrapeptide set forth in co-pending U.S. application Ser. No.09/965,967, which is A-(V/T/I)-(P/A)-(F/Y/I/V) (SEQ ID NO:8).

Without intending to be limited by any explanation as to mechanism, itis believed that the underlying factors influencing binding of thelabeled tetrapeptide AVP-dye to the BIR binding groove include thefollowing:

-   -   1. Recognition is achieved through hydrogen bond interactions        and van der Waals contacts.    -   2. Eight inter- and three intra-molecular hydrogen bonds support        the binding of AVPI in the surface groove on BTR3.    -   3. Three intermolecular contacts between the backbone groups of        Val2/Ile4 in Smac and Gly306/Thr308 in BIR3 allow the formation        of a 4 stranded antiparallel β sheet.    -   4. Ala1 donates 3 hydrogen bonds to Glu314 and Gln319, and its        carbonyl makes contact with Gln319 and Trp323.    -   5. The methyl group of Ala1 fits tightly in a hydrophobic pocket        formed by the side chains of Leu307, Trp310, and Gln319.    -   6. Val2 and Pro3 maintain multiple van der Waals interactions        with Trp323, and Pro3 has an additional interaction with Tyr324.    -   7. The side chain of Ile4 interacts with Leu292, Gly306, Lys297        and Lys299.

Accordingly, the AVP-dye may comprise any suitable detectable label,such as a fluorophore, such that binding of the label does notdetrimentally affect binding of the dye to the BIR3, via any one or moreof the foregoing factors. A particularly suitable dye for use in theAVP-dye is 6-Bromoacetyl-2-dimethylaminonaphthalene (badan) dye. Badanis a fluorogenic dye whose sensitivity to environmental changes haspreviously been made use of to probe protein binding interactions(Boxrud et al. J. Biol. Chem. 275: 14579-14589, 2000; Owenius et al.,Biophys. J. 77: 2237-2250, 1999; Hiratsuka, T. J. Biol. Chem. 274:29156-29163, 1999).

The synthesis of NH₃ ⁺-AVPC(badan)amide is described below, and itschemical structure is shown in FIG. 1. Unless otherwise stated,materials were purchased from Aldrich Chemical Co. (Milwaukee, Wis.) orFisher Scientific (Pittsburgh, Pa.) and used without furtherpurification. Methylbenzhydrylamine (MBHA) solid-phase peptide synthesisresin and Fmoc amino acids were obtained from Advanced ChemTech(Louisville, Ky.) and NovaBiochem (San Diego, Calif.). Badan dye wasobtained from Molecular Probes (Eugene, Oreg.).

The peptide was synthesized on a hand shaker by Fmoc protocol on MBHAresin (Chan, W. C.; White, P. D. Fmoc Solid Phase Peptide Synthesis: APractical Approach; Oxford University Press: Oxford, 2000). The MBHAresin was chosen because the protocol requires that it be stable underboth acidic and basic conditions. The Ala-Val-Pro-Cys peptide wassynthesized using a trityl group to protect the Cysteine thiol. Prior tothe deprotection of the Fmoc group of the alanine, the trityl group wasremoved by the addition of trifluoroacetic acid (TFA), and the cysteinewas derivatized with badan in the presence of diisopropylethylamine(DIEA). The Fmoc group of the alanine was removed with piperidine andthen cleavage from the resin was effected by treatment with anhydrous HFcontaining 10% v/v anisole as scavenger at 0° C. for 45 minutes. Thelabeled peptide was purified by HPLC on a Vydac C18 preparative columnwith gradient elution by solvents A (99% H₂O; 1% CH₃CN; 0.1% TFA) and B(90% CH₃CN; 10% H₂O; 0.1% TFA) and lyophilized to dryness prior toreconstitution in H₂O.

Absorption and emission properties of AVPC-badan are shown in FIG. 2.FIG. 2A shows the absorption and emission spectra of the molecule inwater. FIG. 2B shows the solvatochromicity of AVPC-badan in acetonitrile(ACN), with respect to the emission spectrum. FIG. 3 shows the emissionspectra of AVPC-badan in the presence of BIR3 at differentconcentrations of BIR3.

The aforementioned AVP-dye is used in an assay of test compounds thatmay, like the Smac tetrapeptide AVPI, bind to the BIR3 domain of XIAP,thereby relieving XIAP-mediated suppression of apoptosis. This is ahigh-throughput, cell-free assay, that is assembled as follows. Aprotein comprising the BIR3 domain of an IAP is placed in an assaymedium comprising a suitable buffer, as described above. Preferably,this is a recombinant protein comprising the BIR3 domain, but a full IAPprotein also may be used. An aliquot of the AVP-dye is added to thereaction mixture, in the presence of the test compound. Controlscomprise the BIR3 and the dye in the absence of the test compound and,optionally, BIR3 and the dye in the presence of the naturally occurringtetrapeptide, AVPI. The fluorescence of the reaction mixture at aselected excitation and emission wavelength, e.g., 387 nm excitation,545 nm emission, is measured. Alternatively, a emission spectrum ismeasured at the selected excitation wavelength. In one type ofmeasurement, the test compound is added and an emission spectrum ismeasured by scanning from, e.g., 460-480 nm. In another type ofmeasurement, the emission intensity at a particular wavelength, e.g.,470 nm, is measured. The emission spectrum of the dye bound to BIR3 isdistinctly different from the spectrum of the dye in solution, asdemonstrated in FIGS. 3 and 4. Thus, the binding affinity of the testcompound may be calculated as a function of its ability to displace thedye from the BIR3 domain, according to the following calculation:$K_{relative} = \frac{{{Fraction}_{free}^{2}\lbrack{badan}\rbrack}_{total}}{\left( {1 - {Fraction}_{free}} \right)\left( {\lbrack{AVPX}\rbrack_{total} - {\lbrack{badan}\rbrack_{total}{Fraction}_{free}}} \right)}$

Details of a typical assay are set forth below.

Materials:

-   63 μM BIR3 in 50 mM Kphos buffer pH 7 100 mM NaCl2 mM DTT    -   Four 0.5 ml aliquots of BIR3 stored at −70° C. and thawed over        ice were used-   43.8 μM AVPC-badan in H₂O; chilled to 4° C.    -   absorbance at 387 nm=0.9205; ε_(387nm)=21000 M⁻¹ cm⁻¹-   50 mM tetrapeptide solutions in H₂O; chilled to 4° C.-   50 mM Kphos buffer pH 7 100 mM NaCl 2 mM DTT; chilled to 4° C.-   H₂O (MilliQ purified); chilled to 4° C.    Procedure

Stock solution of badan, BIR3, and buffer were mixed: 2.5 ml of badan,1.75 ml BIR3, and 15.25 ml of buffer were mixed in a glass vial whichhad been chilled to 4° C. Added 390 μL of the stock solution to 50 wellsin the pre-chilled 96 well plate (wells A1-E2).

Stock solution of badan and buffer were mixed: 150 μL badan and 1020 μLof buffer were mixed in a small glass vial (also chilled) and added to 3wells on the plate in 390 μL aliquots (F1-F3).

The 96 well plate was stored over ice in an insulated bucket while theemission spectra of the samples were taken. Fifty μL of the appropriatetest solution (or water, for the control experiments) was added with amicropipet, the solution mixed with a Pasteur pipet before adding thesample to the fluorescence cuvette. While one sample was being scanned,the cuvette from the previous scan was washed with EtOH and then nextsample was prepared.

The PTI fluorometer settings were as follows:

-   λ_(ex)=387 nm; the emission spectrum was scanned from 420-650 nm-   slits=5 nm dispersion-   PMT voltage=750 mV    The scan was done in 1 nm increments and the integration time was 1    s.

Using the above assay, the inventors have screened a wide variety ofpeptides and peptide mimetics for their ability to bind to the BIR3domain of XIAP. As an example, a tetrapeptide library was created, inwhich positions 1, 2 and 4 of the Smac tetrapeptide were substitutedwith other components. In one series of constructions, substitutionswere as follows:

-   -   1. Position 1: XVPI (SEQ ID NO:9), where X=Serine, Glycine or        Aminobutyric acid.    -   2. Position 2: AXPI (SEQ ID NO:10), where X=all twenty naturally        occurring amino acids.    -   3. Position 4: AVPX (SEQ ID NO: 1), where X=all twenty naturally        occurring amino acids.

Samples of results of the assay performed on members of theaforementioned group are shown in Table 1. TABLE 1 SEQ ID: SampleIntensity (470 nm) Fraction_(free) K_(relative) 4 AVPF 16773 0.9741031.5300 11 AVPW 23435 0.94176 23.1330 5 ARPI 29455 0.91253 4.3126 12ALPI 38650 0.86789 3.5812 13 AbuVPI 34770 0.88673 3.0455 14 AIPI 449020.83754 2.6613 15 AVPY 39093 0.86574 2.5442 3 AVPI 54232 0.79224 2.501416 AHPI 41450 0.85430 2.2917 3 AVPI 26924 0.92482 2.2415

The tetrapeptides AVPF (SEQ ID NO:4), AIAY (SEQ ID NO:17) and AVAF (SEQID NO: 18) correspond in sequence to Drosophila homologs of Smac.Results showed that tetrapeptides containing these sequences boundstrongly to BIR3 (AVPF shown in Table 1, other results not shown).

The most successful modification at position 2 was ARPI (SEQ ID NO:5).The positive charge on the arginine residue may have contact with thesurrounding negatively-charged residues in the binding pocket, resultingin the strong binding observed with ARPI (SEQ ID NO:5).

As mentioned, a tetrapeptide library of position-4 modifications wascreated. Table 2 below sets forth binding constants obtained for eachmember of this library, as tested with the assay of the invention. TABLE2 SEQ ID: Tetrapeptide K 4 AVPF >20 3 AVPI (std) 4.2149 15 AVPY 1.169211 AVPW 1.0817 19 AVPL 0.34232 3 AVPI 0.29080 20 AVPD 0.17988 21 AVPT0.14300 2 AVPC 0.10340 22 AVPV 0.10111 23 AVPG 0.089481 24 AVPH 0.07520925 AVPQ 0.066115 26 AVPA 0.055180 27 AVPM 0.052881 28 AVPE 0.037089 29AVPN 0.015724 30 AVPS 0.013041 31 AVPP 0.010695 32 AVPK 0.0070200 33AVPR 0.0014831

Emission spectra of samples from this binding assay are shown in FIG. 4.As can be seen from FIG. 4 and the results set forth in Table 1 andTable 2, the tetrapeptide AVPF (SEQ ID NO: 4) bound strongly to the BIR3domain, as evidenced by its ability to displace the AVP-dye. AVPW (SEQID NO11): and AVPY (SEQ ID NO:15) also showed binding at a strengthequivalent to that of the naturally-occurring Smac peptide, AVPI (SEQ IDNO:3). By contrast, AVPK (SEQ ID NO:32) bound BIR3 only weakly.

In summary, the assay described herein has been demonstrated effectivein identifying compounds that are capable of binding to the BIR3 domainof XIAP. Certain tetrapeptides with greater binding ability than thenaturally-occurring Smac tetrapeptide have been identified. Thesetetrapeptides may be developed as therapeutic agents for the promotionof apoptosis in treatment of diseases or pathological conditions inwhich cell proliferation plays a role. The assay may be further used inhigh throughput screening of large panels of compounds generated bycombinatorial chemistry or other avenues of rational drug design.

The following nonlimiting example is set forth to describe the inventionin greater detail. The example contains data that replicate andsupplement the data presented above. The example also describesadditional tetrapeptide analogs, including N-methyl analogs and a dualsubstituted tetrapeptide, ARPF.

EXAMPLE 1 Molecular Targeting of Inhibitor of Apoptosis Proteins Basedon Small Molecule Mimics of Natural Binding Partners

In this example, a fluorescence assay was used to test the binding of alibrary of tetrapeptides modeled on the Smac N-terminus to the surfacepocket of the BIR3 region of XIAP. The results make it possible to parsethe contribution of each residue of the tetrapeptide to the totalbinding energy of the interaction.

Materials and Methods

Materials. Unless otherwise stated, materials were purchased fromAldrich Chemical Co. (Milwaukee, Wis.) or Fisher Scientific (Pittsburgh,Pa.) and used without further purification. Methylbenzhydrylamine (MBHA)solid-phase peptide synthesis resin, Rink amide resin, and9-Fluorenylmethoxycarbonyl (Fmoc) protected amino acids were obtainedfrom Advanced ChemTech (Louisville, Ky.) and NovaBiochem (San Diego,Calif.). 6-Bromoacetyl-2-dimethylaminonaphthalene (badan) dye wasobtained from Molecular Probes (Eugene, Oreg.).

Synthesis of A VPC-badan. The peptide was synthesized by Fmoc protocolon MBHA resin. The MBHA resin was chosen because the protocol requiresthat the linkage to the solid support be stable under both acidic andbasic conditions. The Ala-Val-Pro-Cys-NH₂ (AVPC; SEQ ID NO:2) peptidewas synthesized using a trityl group to protect the cysteine thiol. Thetrityl group was removed by treatment with trifluoroacetic acid (TFA),and the cysteine was derivatized with badan in the presence ofdiisopropylethylamine (DIEA). The Fmoc group of the alanine was removedwith piperidine and then cleavage from the resin was effected bytreatment with anhydrous HF containing 10% v/v anisole as scavenger at0° C. for 45 minutes. The labeled peptide was purified by HPLC on aVydac C18 preparative column with gradient elution by solvents A (99%H₂O; 1% CH₃CN; 0.1% TFA) and B (90% CH₃CN; 10% H₂O; 0.1% TFA) andlyophilized to dryness prior to reconstitution in H₂O.

Synthesis of N-Fmoc-N-methyl-amino acids. N-methyl-amino acids weresynthesized according to the methods of Freidinger et. al. (J. Org.Chem. 48: 77-81, 1983). The N-Fmoc-N-methyl-isoleucine andN-Fmoc-N-methyl phenylalanine were chromatographed over silica gel (5%methanol in chloroform as eluent); the N-Fmoc-N-methyl-valine was usedwithout further purification.

Synthesis of Tetrapeptide Libraries. With the exception of the positionone library and A(N-Me)VPI, all of the library molecules weresynthesized on an Advanced ChemTech 396 MPS automated peptidesynthesizer by Fmoc protocol on Rink amide resin (Chan & White (2000)Fmoc Solid Phase Synthesis, A Practical Approach; Oxford UniversityPress, Oxford). For the AVPX (SEQ ID NO:1) and the AXPI (SEQ ID NO:10)libraries, the X positions were substituted with all twenty naturallyoccurring amino acids. The side chains of the amino acids that aresensitive to side reactions were protected as follows: cysteine,histidine, asparagine, and glutamine were protected using a tritylgroup; aspartic acid, glutamic acid, serine, threonine, and tyrosinewere t-butyl protected; lysine and tryptophan were protected by Bocgroups; and a pentamethyldihydrobenzofuran group was used to protect thearginine. After the alanine was added, deprotection and cleavage of thetetrapeptides from the resin was effected by adding 1 ml of a 95% TFA,2.5% water, and 2.5% triisopropylsilane (TIS) solution to each well, andshaking for 1 hour. The cleavage solution was collected and a further0.5 ml of the cleavage solution was added to each well and mixed foranother hour. The combined cleavage solutions were added to 20 ml ofwater, lyophilized to dryness, then taken up in 5 ml of water beforebeing filtered through syringe filters (0.2 μ) and lyophilized again.

The position one tetrapeptides and A(N-Me)VPI (SEQ ID NO:34) weresynthesized on a hand shaker, also by Fmoc protocol on Rink amide resin.Cleavage and work up were done as described above. The presence of thedesired tetrapeptide molecules was confirmed by mass spectroscopy.

The tetrapeptides were reconstituted in water and test solutions weremade that were approximately 200 mM in the tetrapeptides. Exactconcentrations were determined for 10 representative test solutions by¹H-NMR using a dioxane solution of known concentration as an externalreference. The concentrations of the other test solutions were taken tobe the average value of the known solutions from the same librarysynthesis.

Expression and Purification of BIR3. Recombinant XIAP-BIR3 (residues238-358) was overexpressed as a GST-fusion protein using pGEX-2T(Amersham Biosciences). The soluble fraction of the GST-BIR3 in the E.coli lysate was purified over a glutathione sepharose column, andfurther purified by anion exchange chromatography (Mono-Q, AmershamBiosciences). The fusion protein was cleaved by thrombin, and the GSTportion was removed by the glutathione sepharose column. The BIR3protein was further purified over a gel filtration column (Superdex 30,Amersham Biosciences).

Fluorescence Experiments. Luminescence spectra were recorded using aPhoton Technologies, Inc. fluorometer with a Xe arc lamp and a PMTdetector. The absorbance of all solutions was less than 0.2 at theexcitation wavelength (387 nm). The buffer used in all of thefluorescence experiments was 50 mM potassium phosphate, 100 mM NaCl, 2mM 1,4-dithio-DL-threitol (DTT), pH 7.

Determination of A VPC-badan binding constant to BIR3. 2 ml of a 2 μMAVPC-badan stock solution (buffer same as above) was titrated with aBIR3 stock solution from 0 to 10 μM in 15 μL increments. Thedissociation constant for AVPC-badan and BIR3 was determined from theintensity observed at 470 μm after each addition of the protein.

Assay of Tetrapeptide Libraries. The samples were prepared in a 96 wellplate lined with glass tubes, to prevent adsorption of the dye toplastic. The plate was stored on ice in the dark between measurements. Asmall volume cuvette, with a path length of 2 mm, was used to collectthe emission spectra. 2.5 ml of a 44 μM aqueous solution of AVPC-badan,1.75 ml of a 63 μM BIR3 solution, and 15.25 ml of buffer were mixed togive a stock solution which was 5.6 μM in both AVPC-badan and BIR3. 390μL of this stock solution were added to 50 wells of the 96 well plate.50 μL of the test tetrapeptide solutions were added and mixedimmediately prior to taking the emission spectra. The final solutionswere 5 μM in both badan and BIR3, and approximately 20-30 μM in thetetrapeptide solutions. 50 μL of water were added to three of the wellsby way of controls, to determine the intensity observed when theAVPC-badan was bound to BIR3. 190 μL of AVPC-badan and 1020 μL of bufferwere mixed and added to three wells in 390 μL aliquots. 50 μL of waterwas added to these wells, again as controls, to determine the intensityof the unbound dye. Equilibrium constants were determined by relatingthe observed intensity of the test solution at 470 nm to the averagevalues obtained from the control experiments.

Results

The binding of various tetrapeptide mimics to the BIR3 domain of XIAPwas determined using a fluorescence-based competition assay. The assayis based on an environment-sensitive fluorogenic dye molecule, badan.Badan is a dye whose sensitivity to environmental changes has previouslybeen used to probe protein binding interactions. A tetrapeptide based onthe Smac binding motif, Ala-Val-Pro-Cys-NH₂ (AVPC; SEQ ID NO:2), wasderivatized with the badan molecule to create a binding interaction withBIR3. When AVPC-badan binds to the surface groove of BIR3, changing theenvironment of the dye from water to the hydrophobic interior of theprotein, the result is a large shift in both fluorescence maximum andintensity. The K_(D) for the AVPC-badan/BIR3 complex, as determined froma fluorescence titration, is 0.31±0.04 μM. The AVPC-badan can bedisplaced from the binding pocket of the protein by any competingmolecule. As the dye is displaced from the binding pocket by the testmolecule, the emission shifts back towards the aquated spectrum. Thus,the observed emission intensity of the dye can be related to the degreeof displacement of AVPC-badan by the test molecules. This allows themost promising inhibitors to be quickly determined, and structuralinformation about effective inhibitors can be incorporated into thedesign of candidates for the next round of testing.

Using the four N-terminal residues of Smac as a starting point, sixlibraries of related tetrapeptides were synthesized (Scheme 1) andevaluated in terms of their ability to displace AVPC-badan from thepeptide binding groove on the surface of BIR3. The tetrapeptidelibraries were designed to deconvolve the contribution of each aminoacid to the binding of Smac to BIR3 (Scheme 1). The position one libraryonly consisted of three members, reflecting the critical role that Ala1plays in the recognition of the binding element by BIR3. The role ofposition three was explored using a tetrapeptide based on the N-terminalsequence of Reaper, one of the few natural binding partners without aproline in position three (Table 3). Libraries of positions two andfour, over all twenty naturally occurring amino acids, were synthesized.The tetrapeptide ARPF (SEQ ID NO:35) was synthesized to investigate thepossibility of additivity by modifying both positions simultaneously.

There are two bonds in the tetrapeptide that are vulnerable toproteolysis; the peptide bond between position one and position two, andthe peptide bond between position three and four. One means of renderingthese bonds more resistant to proteolysis is to replace the hydrogen onthe amide with a methyl group. Several tetrapeptide homologs weresynthesized with N-methyl amino acids to explore the effect suchmodifications have on the affinity of these compounds for BIR3.

The dissociation constants (K_(D)) for the library members are listed inTable 4. The tetrapeptide mimics displace badan from BIR3 with varyingfacility (Table 4, FIG. 6A). The K_(D) values ranged from 0.02 μM togreater than 100 μM. The conservation of sequence of the binding motifobserved across the range of protein binding partners suggests thatnature has optimized the appropriate sequence to some extent, but thevariety of tetrapeptides tested in this assay explores the specificcontribution made at each position to the overall binding interaction.

TABLE 4 K_(D) for Tetrapeptide Homologs (Numbers to the right of eachsequence in parentheses are SEQ ID NOS) K_(D) (μM) Natural Analogs AVPI(3) 0.48 AVPIAQKSE (36) 0.40 AVAF (46) 0.56 AVPF (4) 0.04 AVPY (15) 0.30Position 1 AbuVPI (13) 0.24 GVPI (6) 9 SVPI (47) 27 Position 2 ARPI (5)0.18 ALPI (12) 0.29 AHPI (16) 0.33 AIPI (14) 0.39 AKPI (48) 0.57 AYPI(49) 0.59 ACPI (50) 0.65 AMPI (51) 0.73 AFPI (52) 0.79 AQPI (53) 0.94AWPI (54) 0.99 ATPI (55) 1.2 ASPI (56) 1.4 ANPI (57) 1.5 AEPI (58) 2.7AAPI (59) 2.8 ADPI (60) 17 AGPI (7) 46 APPI (61) >100 Position 4 AVPW(11) 0.11 AVPL (19) 0.49 AVPC (2) 1.4 AVPV (22) 1.5 AVPT (21) 2.1 AVPM(27) 2.3 AVPS (30) 4.4 AVPG (23) 4.7 AVPP (31) 5.7 AVPD (20) 7.3 AVPH(24) 7.3 AVPA (26) 14 AVPK (32) 28 AVPE (28) 93 AVPR (33) >100 AVPN(29) >100 AVPQ (25) >100 Positions 2 and 4 ARPF (35) 0.02 N-methylAnalogs ARP(N-Me)F (62) 0.71 AVP(N-Me)F (63) 0.89 A(N-Me)VPF (64) 83A(N-Me)VP(N-Me)F(65) 91 AVP(N-Me)I (66) 174 ARP(N-Me)I (67) 190A(N-Me)VPI (68) 257

Discussion

Residue 1

In previous studies, it was noted that mutations of the N-terminal aminoacid of Smac completely abrogated the binding interaction between Smacand BIR3. The recognition between Smac and the surface groove of theBIR3 is based on a combination of eight intermolecular hydrogen bondsand van der Waals contacts. The necessity of the N-terminal alanine isobvious from the crystal structure. Ala1 donates three hydrogen bonds tonearby residues in the surface groove of BIR3, and its carbonyl groupmakes two additional contacts. The methyl group of Ala1 fits tightlyinto a hydrophobic pocket, and any modification of the alanine residuemust be carefully designed to avoid steric hindrance in this pocket, ordisruption of any of these essential hydrogen bonds. Although the nextthree residues contribute to the positioning of Ala1 in the bindingpocket, their identity does not appear to be as critical as that of theAla1.

The position one library members demonstrate how sensitive the bindinginteraction is to any modification at this position. Binding is greatlydiminished with GVPI (SEQ ID NO:6), consistent with an earlier report,and SVPI (SEQ ID NO:47) is also a diminished binder, but a slightenhancement in binding was observed with the unnatural amino acid,aminoisobutyric acid (Abu).

Residue 3

AVAF (SEQ ID NO:46) has a binding affinity similar to that observed forthe other natural analogs, AVPI (SEQ ID NO:3) and AVPIAQKSE (SEQ IDNO:36). However, this affinity is diminished by greater than a factor often relative to that observed for the AVPF (SEQ ID NO:4) tetrapeptidefrom the position two library. Previous studies have also noted adecrease in binding affinity when the proline is replaced by alanine.Based on that observation, and the relative homogeneity observed in thenatural binding partners at position three (Table 3), it would seem thatreplacing the proline will diminish the binding affinity of the testtetrapeptide.

Residue 2

As stated earlier, nature has already optimized the appropriate sequenceto some extent. However, the position two library gives some surprisingresults. The high affinity of tetrapeptides such as ARPI (SEQ ID NO:5)and AHPI (SEQ ID NO:16) relative to the natural sequence of AVPI (SEQ IDNO: 3) would seem to indicate that positive charge at position two wouldincrease the binding affinity of the peptide. This is not an unexpectedresult given the negatively charged residues that line the bindingpocket of BIR3. Nonetheless, none of the natural binding partners of IAPlisted in Table 3 has positively charged residues at position two. Allthe natural LAP interacting motifs that have been observed so far allcontain b-branched amino acids at position two, such as valine,threonine, and isoleucine (Table 3). This result indicates that thenatural sequence can be improved upon, and gives a basis for thestructural design of the next set of potential binding partners.

Residue 4

The X-ray structure of Smac binding to BIR3 indicates that there are nointermolecular hydrogen bonds to residue 4, and, of the four residues ofthe binding motif, residue 4 is the least sterically hindered. Thiswould seem to make position four least sensitive to modification.Indeed, the K_(D) that is observed for the AVPC (SEQ ID NO: 2)tetrapeptide (Table 4) is greater than that of the AVPC-badan, whichindicates that binding is slightly enhanced by the presence of the dye.However, a much wider range of K_(D)s is observed for the position fourlibrary than for the position two library. Although modification at thisposition can lead to the greatest enhancement in binding affinity thatis observed, it can also essentially destroy the binding interaction.

The AVPF (SEQ ID NO:4) tetrapeptide was by far the most strongly bindinglibrary member, closely followed by AVPW (SEQ ID NO:11). AVPY (SEQ IDNO:15) was also determined to have a binding affinity slightly greaterthan the natural analog, AVPI (SEQ ID NO:3). These results indicate thatan aromatic group side chain on the amino acid at position foursubstantially enhances the binding affinity of the tetrapeptide forBIR3. This result is consistent with phylogenic data: other proteinsthat interact with LAPs have phenylalanine or tyrosine at position four(Table 3).

When high affinity substitutions at position two and four were probedsimultaneously using the ARPF tetrapeptide, the effects were found to beadditive. Consequently, the detrimental effect on binding affinityobserved with the N-methylated tetrapeptides could be somewhatcounteracted by the increased affinity gained from the appropriatechoice of amino acid.

N-methyl Analogs

N-methylation at the peptide bond between residues 1 and 2 disrupts astructurally defined hydrogen bond, and has a correspondingly largeeffect on binding. By contrast, N-methylation of residue 4 has a muchsmaller effect, consistent with structural data, which show no hydrogenbond to this amide. From a standpoint of molecular design, this relievesan important design constraint. Consideration of side chaincontributions to the free energy of binding, ΔG_(b), using the freeenergy of transfer from ethanol to water, ΔG_(t) (EtOH—H₂O), toapproximate the energy contribution of the side chain for hydrophobicamino acids, follows a clear general trend. More hydrophobic amino acidsclearly bind more strongly, as indicated in FIG. 6B. The obviouscorrelation indicates that there is little specificity of interaction,but also suggests that the full hydrophobic effect is not realized. Forexample, the ΔG_(t) of W is greater than that of F, but the ΔG_(b) ofAVPF (SEQ ID NO:4) is greater than that of AVPW (SEQ ID NO:11). A moredetailed analysis can be obtained by modeling the various peptides ontothe known structure and determining the solvent exposed surface areawithin the model.

This invention is not limited to the embodiments described andexemplified above, but is capable of variation and modification withinthe scope of the appended claims.

1. An assay for determining if a test agent is capable of binding a BIRdomain of an Inhibitor of Apoptosis Protein (IAP), comprising the stepsof: a) providing a detectably labeled peptide or peptidomimetic compoundthat binds to a BIR domain of the IAP, wherein the compound has aformula:R₁—R₂—R₃—R₄ wherein R₁ is A or a mimetic of A; R₂ is V, T or I or amimetic of V, T or I; R₃ is P or A or a mimetic of P or A; and R₄ is anyamino acid or a mimetic thereof and the detectable label is associatedwith R₄; wherein at least one measurable feature of the detectable labelchanges as a function of the labeled compound being either bound to theIAP or free in solution; b) contacting the IAP with the labeled compoundunder conditions enabling binding of the labeled compound to the IAP,thereby forming a labeled compound/IAP complex having the measurablefeature; c) contacting the labeled compound/IAP complex with the testagent; and d) measuring displacement of the labeled compound from thelabeled compound/IAP complex, if any, by the test agent, by measuringthe change in the measurable feature of the labeled compound, therebydetermining if the test agent is capable of binding to the IAP.
 2. Theassay of claim 1, wherein the labeled compound is a peptide AVPX,wherein X is any amino acid
 3. The assay of claim 1, wherein the labelis a fluorigenic dye.
 4. The assay of claim 3, wherein the labeledcompound is a peptide AVPX, wherein X is any amino acid and is directlyor indirectly linked to the fluorigenic dye.
 5. The assay of claim 4,wherein the labeled compound is AVPC-badan dye.
 6. The assay of claim 1,wherein the BIR domain is a BIR3 domain or a BIR2 domain.
 7. The assayof claim 1, wherein the BIR domain is provided as part of an intact IAP.8. A detectably labeled compound for performing a assay to determine ifa test agent is capable of binding a BIR domain of an Inhibitor ofApoptosis Protein (IAP), wherein the compound has a formula:R₁—R₂—R₃—R₄ wherein R₁ is A or a mimetic of A; R₂ is V, T or I or amimetic of V, T or I; R₃ is P or A or a mimetic of P or A; and R₄ is anyamino acid or a mimetic thereof and the detectable label is associatedwith R₄; wherein at least one measurable feature of the detectable labelchanges as a function of the labeled compound being either bound to theIAP or free in solution.
 9. The labeled compound of claim 8, comprisinga peptide AVPX, wherein X is any amino acid
 10. The compound of claim 8,wherein the label is a fluorigenic dye.
 11. The compound of claim 10,comprising a peptide AVPX, wherein X is any amino acid and is directlyor indirectly linked to the fluorigenic dye.
 12. The compound of claim11, which is AVPC-badan dye.
 13. An assay for determining if a testcompound is capable of binding a BIR3 domain of an Inhibitor ofApoptosis Protein (LAP), comprising the steps of: a) providing a labeledmimetic of an AVPI tetrapeptide that binds to the BIR3 domain, whereinat least one measurable feature of the label changes as a function ofthe mimetic being bound to the IAP or free in solution; b) contactingthe IAP with the labeled mimetic under conditions enabling binding ofthe mimetic to the IAP, thereby forming an IAP/labeled mimetic complexhaving the measurable feature; c) contacting the IAP/labeled mimeticcomplex with the test compound; and d) measuring displacement of thelabeled mimetic from the IAP/labeled mimetic complex, if any, by thetest compound, by measuring the change in the measurable feature of thelabeled mimetic, thereby determining if the test compound is capable ofbinding to the IAP.
 14. The assay of claim 13, wherein the labeledmimetic is AVPX, wherein X is directly or indirectly linked to afluorigenic dye.
 15. The assay of claim 13, wherein the labeled mimeticis AVPC-badan dye.
 16. The assay of claim 1, wherein the LAP issubstituted with a portion of the IAP comprising the BIR3 domain.